Many significant protein–DNA interactions involve sharp bending and looping of double-stranded DNA (dsDNA) with curvature radius of 2–20
). A remarkable example is the histone–DNA complex in eukaryotic cells; genomic dsDNA is wrapped around histone complexes with radii of 4.5
nm. The mechanistic properties of the histone–DNA complex is thought to be involved in the control of transcription activity (2
). Most transcription factors also deform DNA by bending or looping DNA strands to regulate gene expression (3
). DNA condensation occurs in extremely small viral capsids (radius of 15–50
nm) and is another example of extensive winding or bending of DNA (4
). Thus, revealing the mechanical properties of dsDNA is crucial for understanding the molecular mechanisms of DNA–protein systems, and has been a focal issue in the physicochemical research on DNA (1
Fundamental aspects of DNA bending mechanics have been studied using biochemical bulk measurements (5
) and by pioneering single-molecule DNA stretching experiments (6
). DNA bending is well described by the worm-like chain (WLC) model (8
), in which the most important parameter is the persistence length, Lp
, which characterizes the filament’s resistance to thermal bending. The persistence length of DNA is reported to be ~50
nm for dsDNA, although the Lp
of DNA changes depending on the experimental conditions and the DNA sequence used.
The single-molecule DNA stretching experiment has high versatility; it offers a unique experimental platform that allows one to study not only the mechanical properties of DNA but also the conformational dynamics of DNA associating proteins at the single-molecule level (10
). Unidirectional motion of RNA polymerase or diffusive translocation of DNA-binding proteins along DNA strand were analyzed to elucidate their working principles.
However, DNA stretching experiments have limitations for the study of the micromechanics of sharply bent DNA or the molecular mechanism of DNA associating protein that induces DNA bending or binds to bent DNA. DNA stretching experiments typically measure the ensemble-averaged stiffness over a long DNA strand, in which small fragments may experience many small bending events influenced by thermal energy. Because thermal energy supplies only low forces in the order of ~0.1 pN (11
), it is rarely possible to induce sharp bending of DNA with a curvature radius in nanometer range in DNA stretch experiments. Therefore, methodology that controls the bending curvature of DNA is required to explore the micromechanics of sharply bent DNA and its physiological role.
Several studies have been carried out to reveal the fundamental mechanical features of sharply bent DNA. Biochemical approaches, including DNA ligase-catalyzed cyclization experiments, have been used to quantitatively measure the ligation efficiency and circularization of short DNA fragments against bending force (12–14
). These experiments allowed the estimation of the flexibility of DNA based on the ratio of the circularly-ligated to linearly-ligated fragments in a wide range of fragment lengths. Atomic force microscopy (AFM) provided more direct measurements of the distribution of dsDNA bending angles, allowing the estimation of the bending energy (15
). However, immobilization of DNA on a surface for AFM imaging may have biased the populations of individual DNA forms. The direct measurement of bending force of dsDNA was also attempted by using a single-stranded DNA as a molecular force sensor, both ends of which were linked to the ends of bent dsDNA (16
). However, the precision of the force determination suffered from the intrinsic noise and non-linearity of single-molecule Förster resonance energy transfer, on which this method relied on for the force estimation. It seems plausible that the WLC model fails to explain several results (12
). Further investigation with regard to this issue is still necessary.
In this study, we developed a novel method to wind individual dsDNA molecules around the rotary motor protein F1
) to directly measure the force and elastic energy required to bend dsDNA at nanometer-scale curvature radii. F1
is the water-soluble portion of the Fo
–ATP synthase (a) (17
). The minimum ATPase-active complex of the F1
motor is the α3
γ subcomplex, in which the γ subunit rotates against the α3
stator in counterclockwise direction upon ATP hydrolysis (19
). As a molecular reel for DNA winding, F1
has an ideal size; the radius of the central shaft of the γ subunit is ~1
nm (radius of α3
stator ring is ~5
nm), which is much smaller than the curvature radius of dsDNA loops induced by histones and transcription factors and thus suitable for winding dsDNA in freely suspended conditions. Our measurements showed that the curvature diameter of wound DNA decreased from 21.4 to 8.8
nm when tension was increased from 0.9 to 6.0 pN. The WLC model with the persistence length of 54
nm well described our data, indicating that tightly bent dsDNA retained its structural integrity.
Figure 1. Experimental setup. (a) The molecular reel was constructed of F1, a magnetic bead, and the Fab fragment on Ni–NTA glass. A biotinylated anti-DIG Fab fragment (orange) specifically linked the γ subunit (red) of F1–ATPase and the (more ...)